I think my brain will explode coz i cant find this answer anywhere!!!

[amywy88]

Can anybody tell me specifically what an in-gel digest is, when talking about trypsin digest of proteins...

......just so I dont have to throw myself off a cliff!!

Thanks!!

[THECORBYLOON]

This may be of help http://en.wikipedia.org/wiki/In-gel_digestion

[theprof]

OK, I'll begin by saying if you've not jumped off that cliff yet, you're sure to do so after I've finished with you!

Right, I'll begin by saying that proteins In the denatured state following SDS-PAGE electrophoresis are susceptible to enzymatic hydrolysis, producing sets of peptides whose sequences can be predicted by simple computer algorithms.

The most common enzyme used for protein digestion is trypsin (which targets Lys and Arg groups). After an overnight incubation, the resulting peptides are recovered first by aspiration and then by washing the gel pieces with dilute formic acid or aqueous acetonitrile. If necessary, the solvent is removed by evaporation. The aqueous solution of peptides is cleaned up by passage over a Zip-tip reverse-phase column. The peptides are bound to the column because of their hydrophobicity, Inorganic electrolytes and neutral materials do not bind and any traces in the column are removed by careful water washing. The peptides are eluted with a small volume of methanol or acetonitrile.

Each member of the set of peptides has a particular molecular weight that is determined by its amino acid composition. The peptide mass fingerprint is characteristic of a given protein. The fingerprint can be compared against a virtual set of peptides from proteins predicted by computers, using proteome databases that are either publicly available or proprietary. Examples are MASCOT, PepMapper and PepSea.

[theprof]

The molecular weights of the peptides are actually measured using a combination of two methods. Matrix-Assisted Laser Desorption Ionization (MALDI) to evaporate the peptides, and time-of-flight (TOF) mass spectrometry to determine their masses, The peptide mixture from a protein spot is mixed with a UV-absorbing matrix in a saturated solution of cyano-4-hydroxycinnamic acid or sinapinic acid in 50% aqueous acetonitrile spotted onto a target plate. The solution is allowed to dry slowly so that crystals of the matrix now containing the peptides are formed. Once in the MALDI-TOF mass spectrometer, each spot in turn is irradiated with short nanosecond UV laser light pulses, thereby evaporating the matrix and dragging peptide ions into the gas phase. The positively charged ions formed in this initial step are focused by an electric field and then injected into the TOF analyzer by applying an accelerating potential (20 kV). The ions `drift' down the flight tube of the TOF analyzer and by timing their arrival at a distal photodiode detector, their `time-of-flight' and hence their mass-to-charge (m/z) ratio can be determined. Since they are singly charged, they take the form of [M+H]+ molecular ions.

Incidentaly, there are now kits available from Sigma-Aldrich and others that contain a complete set of reagents to carry out tryptic digestions on colloidal Coomassie or fluorescent dye-stained protein bands excised from polyacrylamide gels.

Apologies if I've rattled on a bit too much for your needs but Proteomics is a favourite topic of mine. If you need further info, let me know

[amywy88]

Thanks that was really helpful!!